MicroRNA phenotypic screening accomplished using libraries of microRNA mimics may allow the identification and validation of microRNA function in both the pathology of disease and the profiling of responses to therapeutic intervention. This project is focused on the concept that by comparing the results of microRNA over-expression screens from cells that express wild-type KRAS and mutant KRAS, we can not only identify microRNAs involved in context-dependent survival, but also genes and pathways that would be obscured by due to functional redundancy in a conventional siRNA screen. We will focus our attention on microRNAs that influence the viability of colorectal cancer cells. Colorectal cancer (CRC) is currently the third leading cause of fatalities in cancer patients in the United States. Very few effective treatment options are available for CRC, and many vary dramatically in efficacy depending on the genetic status of the tumor. In particular, the presence of mutant KRAS is an early event in CRC. KRAS mutations are found in a significant proportion of CRC tumors, and tumors harboring these mutations are resistant to treatment. In FY12, we first optimized the conditions for the microRNA mimic library screens in 384-well format in CRC cells that express wild-type or mutant KRAS. After extensively optimizing the conditions for the high-throughput screening using control siRNAs and miRNA mimics that are known to reduce viability in a KRAS-independent manner, we transfected CRC cell lines (wild-type or mutant KRAS) with miRNA mimics (from Dharmacon) to specifically over-express 890 miRNAs in each well of a 384-well plate. This approach has enabled us to identify &gt;20 miRNAs that are significantly lethal to KRAS-mutant cells and have very little or no effect on KRAS-wild-type cells. These candidate microRNAs will be further validated in a panel of cell lines that express wild-type or mutant KRAS mutant. We will next investigate the mechanism(s) that contribute to the difference in survival induced by introduction of the specific candidate microRNAs in these cell lines. This approach will also enable us to identify potential microRNAs that may influence the growth and viability of CRC tumors. To investigate this, we will perform xenograft studies in mice upon over-expression of candidate microRNAs in KRAS mutant and KRAS wild-type cells. To further establish the anti-cancer activity of the candidate microRNAs on NSCLC cells, we will also conduct follow-up analysis of these microRNAs in mouse models of CRC cancer. The microRNAs identified in this project will allow us to elucidate vulnerable pathways in KRAS-dependent tumors that can be exploited for a therapeutic or predictive benefit in a typically difficult to manage class of tumor.